Peptide plasminogen activators

ABSTRACT

Novel polypeptide compositions based on the amino acid sequence of tissue plasminogen activator (tPA) are provided having improved properties over natural tissue plasminogen activator. Particularly, enhanced specific activity, reduced response to inhibition by plasminogen activator inhibitor, fibrin stimulation of plasminogenolytic activity and/or enhanced affinity to fibrin surfaces are provided by modifying one or more loci by deletions or substitutions. One or both of the N- or C-termini may be modified.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of Ser. No. 08/381,308, filed Jan.31, 1995, now U.S. Pat. No. 5,656,269, which is a continuation of Ser.No. 06/944,117, filed Dec. 22, 1986, now U.S. Pat. No. 5,501,853, whichis a continuation-in-part of our application Ser. No. 06/812,879 filedDec. 23, 1985, now abandoned, the entire specification of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

Tissue plasminogen activator (tPA) is a serine protease involved withthe dissolution of blood clots. The molecule has a number of distinctregions which display amino acid sequence homology with other naturallyoccurring polypeptides. Beginning at the N-terminus is a regionhomologous to fibronectin. The next region has homology with variousgrowth factors. The growth factor region is followed by two kringleregions. Finally, there is the active site finding analogy with otherserine proteases.

There are at least four different properties associated with tissueplasminogen activator and its ability to lyse blood clots in vivo. Thefirst is the specific activity of the protease function in cleavingplasminogen to produce plasmin which in turn degrades fibrin. The secondproperty is the sensitivity to inhibition by plasminogen activatorinhibitor. The third property is the fibrin dependence of plasminogenactivator for its plasminogenolytic activity. The fourth is the bindingof tPA to fibrin surfaces. Each of these factors plays a role in therapidity and the specificity with which plasminogen activator willcleave plasminogen to plasmin in the presence of blood clots. It wouldtherefore be of substantial interest to be able to produce polypeptideshaving tissue plasminogen activator activity which will provide forimproved properties in one or more of these categories.

BRIEF DESCRIPTION OF THE RELEVANT LITERATURE

The use of mutagenesis to enhance native properties of a naturallyoccurring protein has been reported by Craik et al., Science (1985)228:291; Rosenberg et al., Nature (1984) 312:77-80 and Wilkinson et al.,Nature (1984) 307:187-188. Andreasen et al., EMBO J. (1984) 3:51-56,report that clipping at the cleavage site of tPA is necessary forprotease activity. Ny et al., Proc. Natl. Acad. Sci. USA (1984) 81:5355,report the structure of human tPA and the correlation of introns andexons to functional and structural domains. A. J. van Zonneveld et al.,Abstract from ISTH Meeting, Jul. 14-19, 1985, report on the relationshipbetween structure and function of human tissue-type plasminogenactivator. J. Biol Chem. (1986) 261:14214-14218. Opdenakker et al., EMBOWorkshop on Plasminogen Activation (Amalfi, Italy, Oct. 14-18, 1985)report the effect of removal of carbohydrate side chains on tPAactivity. Loskutoff et al., Proc. Natl. Acad. Sci. USA (1983)80:2956-2960, and Loskutoff, Thrombos. Haemostos, THHADQ, 54(1):118(S699) describe the properties of plasminogen activator-inhibitor.

SUMMARY OF THE INVENTION

Polypeptides having plasminogen activator activity are provided byemploying deletions or substitutions, individually or in combination inthe tPA molecule, where glycosylation is changed, the C-terminustruncated and the cleavage site modified. The resulting products finduse in lysis of fibrin clots and prevention of blood clot formation byactivating plasminogen. These polypeptides may be produced by expressionof mutated genes created by site-directed mutagenesis.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Novel polypeptides having enhanced properties as plasminogen activatorsare provided including DNA coding sequences coding for suchpolypeptides, expression cassettes containing such sequences forexpression in an appropriate host, hosts capable of expressing the novelpolypeptides and methods for producing the novel polypeptides. The novelpolypeptides involve changes at at least one glycosylation site, atleast one change at the cleavage site in the region from 272 to 280,particularly in the sequence Phe₂₇₄ -Arg₂₇₅ -Ile₂₇₆ -Lys₂₇₇ (FRIK),and/or truncation at one or both termini of the molecule particularlythe C-terminus. Where the only modification is at the glycosylationsites, both glycosylation sites at 117-119 and 184-186 will be modified,either the same or different. (The numbering of the amino acids which isemployed is based on that reported by Pennica et al., Nature (1983)301:214-221, beginning at serine in the sequenceglycine-alanine-arginine-serine-tyrosine.) By introducing these changesin polypeptides having tissue plasminogen activator activity and derivedfrom human tissue plasminogen activator improved physiologicalproperties can be achieved.

The novel polypeptides exhibit enhanced proteolytic and specificallyplasminogenolytic activity, reduced sensitivity to plasminogen activatorinhibition, increased affinity for fibrin and/or increased fibrindependence for plasminogenolytic activity.

The first change of interest is the modification of at least oneglycosylation site at amino acids 117-119, 184-186 and 448-450, whereeither the asparagine is changed, conservatively or non-conservatively,or the serine or threonine is changed non-conservatively, particularlyby gly, ala or val. Of particular interest are the glycosylation sitesat 117-119 and 184-186. Mutations of interest include substitutingasparagine with other amino acids, preferably glutamine, valine,leucine, isoleucine, alanine or glycine, more preferably glutamine orsubstituting serine or threonine with other amino acids, preferablyalanine, valine, or methionine, that is, aliphatic amino acids of from 3to 5 carbon atoms lacking mercapto or neutral hydroxyl substituents.Substituting asparagine with glutamine at the glycosylation sites at 117and 184 is of special interest.

Depending upon the desired change in the properties of the mutatedproduct, either conservative or non-conservative changes may beinvolved. Conservative changes are indicated by the amino acids includedwithin a semi-colon. G, A; V, I, L; D, E; K, R; N, Q; S, T; and F, W, Y;where an amino acid in one grouping is substited by an amino acid inanother grouping or an amino acid which is not indicated above, suchchange will be considered non-conservative.

It is found that specific activity can be enhanced by reducing theamount of glycosylation of the molecule, particularly at theglycosylation sites in the kringle structures, more particularly thefirst two glycosylation sites proceeding from the N- to the C-terminus.

Various lesions may be introduced at the glycosylation sites, such asdeletions, substitutions, insertions, or the like, to change theglycosylation site triad so as to destroy the glycosylation site. Forexample, glycosylation sites at 117-119 and 184-186 and 448-450 may bemodified, either the same or differently.

The next area of interest is modification of the cleavage site whichoccurs at amino acids 274 to 278. Of particular interest areconservative changes, such as substituting lysine at 277 with arginine.Other substitutions include replacing isoleucine at 276 with valine,leucine, glycine or alanine. It is found that a specific modification atthe cleavage site can reduce sensitivity to plasminogen activatorinhibition, so that in vivo, the polypeptide will have an increasedactivity as compared to the wild-type tissue plasminogen activator inthe presence of a naturally inhibiting amount of plasminogen activatorinhibitor.

The third change of interest is truncation of the N- or C-terminus,particularly the C-terminus by at least 1 amino acid and up to 25 aminoacids, usually up to 10 amino acids, more usually up to 3 amino acids.The N-terminus may be truncated by from 1 to 5 amino acids, startingwith ser, followed by tyr, more particularly from 1 to 3 amino acids.

Truncation at the C-terminus provides for enhanced fibrin dependence, sothat greater specificity of the tissue plasminogen activator may beachieved. By increasing fibrin dependence, the amount of plasminogenwhich is cleaved to plasmin away from a clot will be substantiallyreduced, avoiding side effects of the plasmin.

The novel polypeptides of the invention may be produced by combiningtwo, three or more of the changes described above. For example, inaddition to substituting aspargine with glutamine at the glycosylationsites at 117 and 184, an example of the first type of change, thecleavage site at 277 may be modified by substituting lysine witharginine (an example of the second type of change) or the sequence Met525 to Pro 527 may be truncated (an example of the third type ofchange).

In some instances, rather than truncating the tPA the chain may beextended or terminal amino acids extended, generally from about 1 to 12amino acids at one or both of the N- and C-termini. Added amino acidsmay serve as linkers, provide a particular immune response, modify thephysical properties of the molecule, or the like.

The DNA sequences encoding for the subject polypeptides may be preparedin a variety of ways, using chromosomal DNA including introns, cDNA,synthetic DNA, or combinations thereof. The desired mutations may beachieved by cloning the gene in an appropriate bacterial host using asingle-stranded bacteriophage vector or double stranded plasmid vectorand then using a mismatched sequence substantially complementary to thewild-type sequence but including the desired mutations, such astransversions, transitions, deletions, or insertions. Alternatively,where convenient restriction sites exist, one could excise a particularsequence and introduce a synthetic sequence containing the mutations.Other techniques which may be employed are partial exonucleolyticdigestion of a double-stranded template or in vitro heteroduplexformation to expose partially single-stranded regions foroligonucleotide-directed mutagenesis.

Of particular interest is oligonucleotide directed mutagenesis. (Zollerand Smith, Methods Enzymol. (1983) 100:468-500.)

Appropriate oligonucleotide sequences containing the mismatches,deletions or insertions are prepared generally having about 20 to 100nucleotides (nt), more usually 20 to 60 nt. The sequences can be readilyprepared by synthesis and be cloned in an appropriate cloning vector.The oligonucleotides are annealed to a purified template of the tPA geneor tPA analog for site directed mutagenesis. The mutations may beintroduced individually, where the sites of mutation are separated byabout 10 base pairs (bp), more usually by about 20 bp. Mutations atdifferent sites may be introduced in a single step or successive steps,after each mutation establishing the existence of the mutation and itseffect on the activity of the tPA analog.

In designing mutagenesis primers, a number of considerations areinvolved. Usually, the length of the primer is chosen to yield less thanabout 95% complementarity with a target template but greater than about85%. This provides an additional advantage in permitting facile plaquescreening with a mutagenesis primer. Where mutagenesis primers generatedeletions, normally there will be at least about 20 base paircomplementarity on each side of the deletion. Also, the primer shouldprovide approximately equal melting temperatures on either side of thelocus of mutation. There is the further consideration that primersshould be selected so as to minimize, if not eliminate, priming eventsat inappropriate sites on the template. In effect, complementarityshould be less than 70%, preferably less than 60%, at any site otherthan the mutagenesis locus. Thus must be considered not only for thegene of interest, but also for the cloning vector. Of particular concernis complementarity at the 3' end of the primer.

Conveniently, the vector will be a single-stranded circular DNA virus,e.g., M13, particularly where modified for purposes of use as a vector.A convenient vector virus is M13mp9. The mutagenesis primer is combinedwith the template and vector under annealing conditions in the presenceof an appropriate polymerase lacking correction capability, such as theKlenow fragment or T4 DNA polymerase in the presence of the necessarynucleotides and T4 DNA ligase, whereby the cyclic, gene carrying vectoris replicative to provide for the double-stranded replication form. Thedouble-stranded DNA may then be introduced into an appropriate cell. Byproviding for a lytic vector, plaques are obtained which may be screenedwith a primer for the presence of the mutagenized tPA or tPA analog.Usually, at least two repetitive screenings will be required, employingincreased sensitivity so as to enhance the probability of having thecorrect sequence for the mutagenized product.

The putative positive clones may then be purified, e.g., plaquepurified, electrophoretically purified, or the like, and then sequenced.

For sequencing, sequencing primers are designed which will be at leastabout 20 to 30 bases in length and will anneal at a position at leastabout 55 bases away from the site of the mutation locus. In order tohave reasonable assurance of the correct sequence, it is desirable thata region be sequenced which includes at least about 50 bases either sideof the mutation locus.

Once the correctly mutagenized gene is obtained, it may be isolated andinserted into an appropriate expression vector for expression in anappropriate host.

A wide variety of expression vectors exist for use in mammalian, insectand avian hosts. See, for example, those described in EPA 0,092,182,which is incorporated herein by reference. Of particular interest aremammalian expression vectors, which employ viral replicons, such assimian virus, bovine papilloma virus, adenovirus, or the like.

The transcriptional and translational initiation and terminationregulatory regions may be the regions naturally associated with the tPAgene or regions derived from viral structural genes or regions derivedfrom host genes. A large number of promoters are available, such as theSV40 early and late promoters, adenovirus major late promoter, β-actinpromoter, human CMV Immediate Early [IE1] promoter, etc. Desirably, thepromoters will be used with enhancers to provide for improvedexpression.

In many instances it will be desirable to amplify the tPA expressionconstruct. For amplification or for other reasons, it may be desirableto have the expression construct integrated into the genome. Toward thisend the construct will either be joined to an unstable replicationsystem, e.g., yeast ars1, or lack a replication system. For integration,see, for example, Axel and Wigler, U.S. Pat. No. 4,399,216. Usually amarker expression construct will be in tandem with the subjectexpression construct, which marker may be the same or different from theamplifying gene. This can be achieved by having the tPA in tandem withan expression construct, functional in the same host as the tPAexpression construct, and encoding such amplifiable genes as DHFR(dihydrofolate reductase), TK (thymidine kinase), MT-I and -II(metallothionein, e.g. human), ODC (ornithine decarboxylase). Bystressing the host in accordance with the amplifiable gene, e.g.,methotrexate with DHFR, the subject expression constructs may beamplified.

Depending upon the particular hosts and signal sequences used, the tPAanalog which is expressed may be retained in the host cell in thecytoplasm or be secreted. Where retained in the cytoplasm, the tPAanalog may be isolated by lysis of the host cell using standard methodsand purified by extraction and purification, preferably by employingelectrophoresis, chromatography, or the like. Where the tPA is secreted,the secreted tPA may be isolated from the supernatant in accordance withconventional techniques including affinity chromatography.

The combined properties of the polypeptides employed in this inventionare superior to naturally occurring plasminogen activators like tPA.These polypeptides will have at least 0.6 times, often at least 0.7times, usually at least 1.0 times, generally from about 1.1 to 7.0times, and preferably from about 6.2 to 7.0 times the specific activityof natural tPA. The fibrin dependence of the activity will be at least0.4, often at least about 0.6 times, generally at least about 0.7 times,and preferably at least about 1 to 2.5 times, particularly 1.3 to 2.5times the naturally occurring tPA. Susceptibility to plasminogenactivator inhibitor will usually be not greater than natural tPA,preferably about 5% less, based on the sensitivity of the natural tPA,more preferably from about 10% less ranging up to about 90% less, andparticularly about 25% to 90% less than natural tPA. The subjectcompounds will usually be superior to wild-type tPA in at least oneaspect. (See the Experimental section for test procedures.)

Of particular interest are polypeptides having tissue plasminogenactivity, having substantially the same amino sequence as human tPA(fewer than 10 number %, usually fewer than 5 number % differencesincluding substitutions, deletions and insertions). These polypeptideswill have at least one of: (a) specific activity at least about 0.6times, often at least about 0.7 times, usually at least about 1.0 times,generally from about 1.1 to 7.0 times, and preferable from about 6.2 to7.0 times the specific activity of natural tPA; (b) fibrin dependence atleast 0.4 times, often at least about 0.6 times, generally at leastabout 0.7 times, and preferably at least about 1.0 to 2.5 times,particularly about 1.5 to 2.5 times the naturally occurring tPA; (c)reduced susceptibility to inhibition by human plasminogen activatorinhibitor of preferably about 5%, compared to the sensitivity of naturaltPA, more preferably from about 10% less ranging up to about 90% less,and particularly about 25% to 90% less than natural tPA.

The subject polypeptides may be used in a variety of ways in theprophylactic or therapeutic treatment for various vascular conditions ordiseases specifically to protect a mammalian, particularly a human, hostfrom thrombus formation. It may therefore be administered duringoperations where the host may be susceptible to blood clotting, or fortreatment of thrombotic patients, to dissolve blood clots which may haveformed in deep vein thrombosis, pulmonary embolism, cranial and coronarythrombosis.

The subject compounds may be administered enterally or parenterally,especially by injection or infusion. These compounds may be administeredin an effective amount in a physiologically acceptable carrier, such aswater, saline, or appropriate buffer systems in the presence of proteinstabilizing substances selected from the group of gelatin, gelatinderivatives, protective proteins such as albumin, sugar, sugar alcohol,or amino acids solely or in combination with other therapeutic drugs,especially for vascular diseases.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Materials and Methods

Mutagenesis was achieved employing the procedure described by Zoller andSmith, Meth. Enzymol. (1983) 100:468-500, modified as described below.Synthetic DNA mutagenesis and sequencing primers were prepared byautomated oligonucleotide synthesis on a silica support as described byUrdea et al., Proc. Natl. Acad. Sci. USA (1983) 80:7461-7465, usingN,N-diisopropyl phosphoramidities. Sequencing primers were designed forsequencing by the dideoxynucleotide chain termination method inbacteriophage M13 (F. Sanger, S. Nicklen and A. R. Coulson, Proc. Natl.Acad. Sci. USA (1977) 74:5463). The sequencing primers were designed tobe complementary to M13mp9 recombinant templates, generally of 20 to 30bases in length and to anneal at a position at least 55 bases away fromthe mutation locus. The following Table indicates the oligomers employedfor mutagenesis and sequencing.

                  TABLE 1                                                         ______________________________________                                             Mutagenesis primers                                                        1a. CGCGTGCTCTGCCAGTTGGT                                                       - 1b. CTGACCCCTGCCCAAAGTAGC                                                   - 2. TTGAGGAGTCGGGTGTTCCTGGTCA/GGTGTCACGAA--                                  -  TCCAGTCTAGGTAG                                                             - 3. AGAGCCCTCCTCTGATGCGA                                                     - 4. CTCTGATTTTAAACTGAGGCTGGCTGTA                                             - 5. GACTGTTCTCTGAAGTAAATG                                                    -  Note: N denotes substitution from wild type                                     / denotes deletion                                                                   Effect of mutation                                               1. Asn 117 → Gln, Asn 184 → Gln                                  - 2. Delete Met 525 through Pro 527                                           - 3. Lys 277 → Arg                                                     - 4. Lys 277 → Arg, Arg 275 → Lys                               - 5. Asn 177 → Gln, Asn 184 → Gln, Asn 448 → Gln                       -  Sequence primers                                            1a. ACCTTGCCTATCAGGATCAT                                                       - 1b. CGATTCGCCCTGGCAGGCGTC                                                   - 2. GTGGGTCTGGAGAAGTCTGTA                                                    - 3,4. GCACAGGAACCGCTCTCCGGG                                                  - 5. TCGATCTGGGTTTCTGCAGTAGTTGTGGTT                                        ______________________________________                                    

Mutagenesis of M13/tPA recombinants was performed using purifiedtemplates by annealing and extending the appropriate primer with theKlenow fragment or T4 polymerase. Mutant 1 was obtained utilizing bothprimers 1a and 1b in a single round of mutagenesis. Mutant 4 wasobtained using a template derived from mutant 3 in the presence of 125μg/ml gene 32 protein and a helper primer which binds to M13 sequencesadjacent to the insert. The effect of primer 4 (Arg 275→Lys) wascombined with the effect of primer 3 (Lys 277→Arg) to produce the twomutation sites shown above. Mutant 5 was obtained using a templatederived from mutant 1. The effect of primers 1a and 1b (Asn 117→Gln, Asn184→Gln) was combined with the effect of primer 5 (Asn 448→Gln) toproduce the three mutation sites shown above. Following transfection ofJM101 cells (Zoller and Smith, supra), plaques were grown at a densityof 200-1000/plate and were lifted onto filters and screened byhybridization with the appropriate mutagenesis primer or probe. Ten to40 percent of these plaques were initially identified as putativepositive candidates. Rescreening of approximately six of these putativesfrom each experiment by dot blot hybridization of phage yielded strongcandidates. These phage were plaque purified by replating at lowdensity, transferring onto nitrocellulose filters and rehybridizing withprimers. The DNA sequence of putative positive clones was determinedusing suitable primers and template preparations.

Once the mutagenized locus and flanking segments (i.e., at least 50bases) were confirmed by DNA sequence analysis, replicative form (RF)DNAs were digested with SalI restriction endonuclease and inserted intothe mammalian expression vector pSV7d previously digested with SalI andtreated with alkaline phosphatase. The vector provides an SV40 earlypromoter and enhancer for expression of the tPA cDNA, an SV40polyadenylation site, and an SV40 origin of replication for use of thevector in COS cells.

The plasmid pSV7d was constructed as follows: the 400 bp BamHI/HindIIIfragment containing the SV40 origin of replication and early promoterwas excised from pSVgtI (Mulligan, R. et al., J. Mol. Cell Biol.1:854-864 (1981)) and purified. The 240 bp SV40 BclI/BamHI fragmentcontaining the SV40 poly A addition site was excised from pSV2/dhfr(Subramani et al., J. Mol. Cell Biol. 1:584-864 (1981)) and purified.The fragments were fused through the following linker:

                       Stop Codons                                                                       1   2   3                                                5'-AGCTAGATCTCCCGGGTCTAGATAAGTAAT-3'                                                 TCTAGAGGGCCCAGATCTATTCATTACTAG                                         HindIII  BglII SmaI  XbaI       BclI overhang.                          

This linker contains five restriction sites, as well as stop codons inall three reading frames. The resulting 670 bp fragment containing theSV40 origin of replication, the SV40 early promoter, the polylinker withstop codons and the SV40 polyadenylation site) was cloned into the BamHIsite of pML, a pBR322 derivative with about 1.5 kb deletion (Lusky andBotchan, Cell 36:391 (1984)), to yield pSV6. The EcoRI and EcoRV sitesin the pML sequences of pSV6 were eliminated by digestion with EcoRI andEcoRV, treated with Bal31 nuclease to remove about 200 bp on each end,and finally religated to yield pSV7a. The Bal31 resection alsoeliminated one BamHI restriction site flanking the SV40 region,approximately 200 bp away from the EcoRV site. To eliminate the secondBamHI site flanking the SV40 region, pSV7a was digested with NruI, whichcuts in the pML sequence upstream from the origin of replication. Thiswas recircularized by blunt end ligation to yield pSV7b.

pSV7c and pSV7d represent successive polylinker replacements. Firstly,pSV7b was digested with StuI and XbaI. Then, the following linker wasligated into the vector to yield pSV7c:

       BglII EcoRI   SmaI   KpnI XbaI                                               5'-AGATCTCGAATTCCCCGGGGGTACCT                                                    TCTAGAGCTTAAGGGGCCCCCATGGAGATC                                       

Thereafter, pSV7c was digested with BglII and XbaI, and then ligatedwith the following linker to yield pSV7d:

     BglII EcoRI   SmaI XbaI  BamHI SalI                                            5'-GATCTCGAATTCCCCGGGTCTAGAGGATCCGTCGAC                                              AGCTTAAGGGGCCCAGATCTCCTAGGCACGTGGATC                             

The appropriate orientation of the cloned fragments within the vectorwas deduced by restriction site analysis. The mutations were reconfirmedby restriction analysis for SalI and EcoRI and by Southern blotting ofsuch fragments using appropriate, highly specific probes directed at thelocus of the mutation.

Combination Mutants

Four combination mutants: mutant 6 which contains both mutations 1 and2, and mutant 7 which contains both mutations 1 and 3, mutant 8 whichcontains both mutations 2 and 3, and mutant 9 contains all threemutations 1, 2, and 3, were constructed using recombinant DNAtechnology. Each of the mutations is separated by convenient restrictionenconuclease sites which facilitated the assembly of the desiredmutations from fragments of the previously isolated mutants 1, 2, and 3.The specific strategy employed in the construction of each mutant isdescribed below.

Large scale plasmid preparations were carried out for all of the mutantconstructions described. The DNA was used for transfections into tissueculture cells.

Mutant 6 (1&2)

The 1.72 kilobase (kb) BamHI to BstEII fragment of mutant 1 containsboth of the mutated sites Asn 117→Gln and Asn₁₈₄ →Gln

This fragment was ligated to the 3.38 kb BamHI to BstEII fragment ofmutant 2 which contains the desired mutation, the deletion of Met₅₂₅through Pro₅₂₇. This ligation mixture was used to transform competent E.coli. A few of the resultant ampicillin resistant colonies were grown upand their DNA was screened by size and by DNA hybridization analysis ofSouthern blots of the DNA with ³² P end labeled oligonucleotide probesspecific to each mutated region. Thus clones were scored as positive ifthey hybridized to each of the two individual mutant 1 regionoligonucleotides and the mutant 2 specific probe. This screening methodwas used for each of the constructs with the substitution of theappropriate oligonucleotide probe(s) for each new construction. Thespecific oligonucleotides utilized are listed previously.

Mutant 7 (1&3)

The 2.95 kb ScaI fragment of mutant 1 which contains both of the mutatedsites, was ligated to the 2.15 kb ScaI fragment of mutant 3 whichcontains the change Lys₂₇₇ →Arg. Screening of the new construction wasaccomplished using oligonucleotides specific to the mutant 3 change andto each of the mutant 1 alterations.

Mutant 8 (2&3)

The 3.38 kb BamHI to BstEII fragment of mutant 2 containing the desiredalteration was ligated to the 1.72 kb BamHI to BstEII fragment of mutant3, containing the Lys₂₇₇ →Arg mutation. Screening for the properrecombinants was accomplished using oligonucleotides specific to eachmutation.

Mutant 9 (1&2&3)

The strategy for making the triple mutant involved using one of thenewly constructed double mutants, mutant 7. This plasmid was cleavedwith BamHI and BstEII, and the 1.72 kb fragment containing bothmutations was isolated. Plasmid DNA from mutant 2 was digested with thesame restriction enzymes and the 3.38 kb piece (containing the mutation)was isolated.

These two fragments were ligated, and the ligation mix was used totransform competent E. coli. Screening for the correct triple mutantswas done as described above, using oligonucleotides specific to eachmutated site in the DNA.

Mammalian Cell Transfections

COS-7 cells (Gluzman, Cell (1981) 23:175) were transfected with thepSV7d tPA expression plasmids using a modification of the proceduredescribed by Graham and van der Eb, Virology (1973) 52:456-467. Thesamples were added to the dishes in duplicate and allowed to settle ontothe cells for 6 h in a carbon dioxide incubator (37° C.). Six hourslater, the supernatants were aspirated, the cells rinsed gently with Ca-and Mg-free phosphate-buffered saline (PBS-CMF), and the dishes exposedto 15% glycerol as an adjuvant for 3.5-4 min. After rinsing and feedingwith DMEM medium, supplemented with 4.5 mg/ml glucose, 3.7 mg/ml Na₂Co₃, 292 μg/ml glutamine, 110 μg/ml sodium pyruvate, 100 U/mlpenicillin, 100 U/ml streptomycin and 10% fetal calf serum (FCS), themedium was replaced with medium lacking the fetal calf serum. Twelvehours after serum withdrawal, cells were assayed for expression of tPA,using the casein-plasminogen-agar overlay described below. Maximumexpression was observed between 36 and 48 h after the start of thetransfection.

CHO dhfr⁻ cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA (1980)77:4216) were plated at a density of 5×10⁵ to 10⁶ cells per 10 cm dishthe day prior to transfection in nutrient medium (F12 supplemented with1.18 mg/ml Na₂ CO₃, 292 μg/ml glutamine, 100 μg/ml sodium pyruvate, 100U/ml penicillin, 100 U/ml streptomycin, 150 μg/ml proline and 10% FCS).The above-described transfection procedure was employed with themodification that the tPA expression plasmid was mixed with a plasmidbearing as a selectable marker, a dhfr gene driven by the adenovirusmajor late promoter, followed by co-precipitation of the plasmids incalcium phosphate. The plasmid bearing the dhfr gene was constructed byfusing the major late promoter from adenovirus-2 (Ad-MLP, map units16-17.3) to the mouse dhfr cDNA at the 5' end. DNA coding for the intronfor SV40 small t antigen and the SV40 early region polyadenylation sitewas obtained from pSV2-neo (Southern and Berg, J. Mol. Appl. Genet.(1982) 1:327-341), and fused to the 3' end of the dhfr cDNA. These threesegments were subcloned into pBR322 to obtain the plasmid Ad-dhfr.Forty-eight hours after the addition of DNA to the cells, the cells weresplit 1:20 into selective medium (DMEM supplemented with proline andfetal calf serum as above, or with dialyzed fetal calf serum). Aftergrowth in selective medium for 1-2 weeks, colonies appeared and wereassayed for production of tPA by the casein-plasminogen-agar overlayassay and in casein-plasminogen plates for quantitation.

Cell lines from each mutant were grown to confluency in 200 cm² dishesand incubated for 24 h periods in serum-free medium (DMEM), andsupernatants were processed as described below to purify thepolypeptides with plasminogen activator activity.

The following procedures were employed for determining the activity ofthe nine polypeptides described above.

In order to determine the specific activity of the mutant proteins, itwas necessary to quantify the antigen concentration of the samples. AnEIA-kit using polyclonal antibodies (American Diagnostica, Inc.,Greenwich, Conn.) was employed. The test was performed strictly underthe conditions described in the protocol provided with the kit and fivedilutions of each sample were analyzed. The results were expressed asthe average of at least three measurable dilutions.

Concentration of Mutant Proteins with Plasminogen Activivator Activity

All cell culture supernatants were made 0.01% Tween 80™ and 0.01%Na-azide. The supernatants (50 ml) were then incubated for 30 min atroom temperature in the presence of 3 ml Heparin-Sepharose®. TheHeparin-Sepharose suspensions were filled into columns and extensivelywashed with 20 ml of a buffer containing 0.1 M phosphate, 0.01% Tween80™, pH 7.5. Finally, the tPA activity was eluted with a buffercontaining 0.05 M tris-HCl, 1 M KSCN and 0.01% Tween 80™, pH 7.5. Theeluates were then dialyzed at 4° C. for 16 h against 5 L of aphosphate-buffer (0.05 M, pH 7.5) containing 0.01% Tween 80™ and 0.01%Na-azide. The tPA samples were frozen in 1 ml portions at -25° C.

Clot Lysis Test (CLT)

The clot lysis test was performed using a "KC 10-Koagulometer" (Amelung,FRG) which has been modified in order to record the lysis time insteadof the coagulation time.

All reagents were preincubated at 37° C. The tPA standard, thepolypeptide test samples and fibrinogen were diluted to the requiredconcentrations in a phosphate-buffer (0.67 M) containing 1% Haemaccel™(pH 7.4). 0.2 ml of the tPA standard or of the samples were mixed with0.2 ml human plasminogen (10 CTA/ml), 0.5 ml bovine fibrinogen (0.15%)and finally 0.1 ml thrombin (10 IU/ml). The lysis time was recorded andexpressed in tPA units using a calibration curve. The activity of eachmutant protein was analyzed at least three times, and the average wascalculated. Four dilutions of the tPA standard were run with each of thesamples to be analyzed.

Plasminogenolytic Assay

The plasminogenolytic activity of the mutant proteins was analyzed usingthe parabolic assay described by Ranby et al., Thromb. Res. (1982)27:743-749. All reagents were preincubated at 37° C. 0.1 ml of the tPAstandard or of the tPA samples were mixed with 0.5 ml buffer (0.1 Mtris-HCl, pH 7.5 containing 0.1% Tween 80™ and 0.01% Na-azide), 0.1 mlsubstrate (S 2251 (H-D-Val-Leu-Lys-(para-nitroanilide) (Kabi AB,Sweden)), 3 mM), 0.1 ml human plasminogen (1 CTA/ml) and 0.1 ml fibrindegradation products (10 mg/ml). Finally, the mixture was incubated for45 min at 37° C. The reaction was stopped by addition of 0.1 ml aceticacid (50%) and the optical density was read at 405 nm. The calibrationcurve obtained was used to convert the optical density of the samples inunits. The results were expressed as the average of at least fivemeasurable dilutions.

Additionally, the same test was performed in the absence of fibrindegradation products (FDP). In this case, 0.6 ml buffer were requiredinstead of 0.5 ml.

Fibrin Dependence of the Activity

The influence of the co-factor--fibrin or fibrin degradation products(FDP)--on the ability of tPA and mutant polypeptides to activateplasminogen into plasmin was expressed as the ratio between the activityobtained in the clot lysis test or in the fibrin-dependentplasminogenolytic assay and the activity determined in thefibrin-independent plasminogenolytic assay.

Fibrin-Affinity Preparation of Fibrin Clots

0.2 ml aliquots of human fibrinogen (0.3%) were disposed in plastictubes and mixed with 0.005 ml bovine thrombin (500 IU/ml). The mixtureswere allowed to stay for 30 min at 37° C. After completion of thereaction the clots were carefully removed and incubated for 30 min at37° C. in 2 ml buffer (0.1 M tris-HCl), pH 8.0) containing antithrombinIII (3 IU/ml) and heparin (40 IU/ml). Finally, the clots were removedand dialyzed twice for 20 min against 50 ml buffer (0.05 M tris-HCl, 0.1M NaCl, pH 7.5). Before use, the clots were freed of surroundingsolution.

Test Procedure

All samples were first diluted to 200 ng/ml in a Tris-buffer (0.1 M, pH7.5) containing 0.1% Tween 80™. Afterwards the samples were diluted to afinal concentration of 100 ng/ml in the same buffer containing 5% humanalbumin. Subsequently, 0.4 ml of the samples were incubated for 10 minat room temperature in the presence of one fibrin clot. Finally, thesolution was removed and the antigen concentration in the fluid phasewas determined with EIA. This experiment was performed twice for eachsample.

Inhibition Test

The samples were diluted in a Tris-buffer (0.1 M, pH 7.5) containing0.1% Tween 80™ to a final concentration of 100 ng/ml.

0.2 ml aliquots were then mixed with 0.2 ml of a solution containing theplasminogen activator inhibitor at a concentration of 65 urokinaseinhibitory units/ml. In control experiments the polypeptide samplesdescribed above were mixed with 0.2 ml buffer (100%). The mixtures wereincubated for 10 min at 37° C. At the end of the incubation time, thesamples were rapidly diluted and the residual activity analyzed with thefibrin-dependent plasminogenolytic assay. The inhibition test wasperformed twice for each sample. The inhibition susceptibility isexpressed as the percent activity that is inhibited.

The plasminogen activator inhibitor has been purified from humanplacenta. The inhibitory fraction does not contain any proteolyticactivity nor plasmin, thrombin or trypsin inhibitory activity.

The values from the above described assays for the mutant proteins arelisted in Table II.

                                      TABLE II                                    __________________________________________________________________________    Biochemical characterization of the mutants                                        Specific activity            Fibrin dependence                             10.sup.3 U/mg  of the activity                                                Mutant CLT.sup.(1)                                                                                                               1  F-Plg..sup.(3)                                                             2  Plg..sup.(4)                                                               3    R3##                                                                     4    R4##                                                                     5  Inhibitor                                                                  susceptibility (%)                                                            Binding to fibrin        __________________________________________________________________________                                                         (%)                      1    1118                                                                              2.74 1215                                                                              2.87   493 2.13 1.30 1.32  26.1    36                         2 404 0.99 307 0.73 81 0.35 2.77 2.00 35.2 39                                 3 288 0.71 284 0.67 257 1.11 0.61 0.58 12.1 33                                4 232 0.57 214 0.51 169 0.73 0.78 0.68 17.7 34                                5 NA 3.2 NA 2.8 NA 7.4 0.43 0.38 20.3 46                                      6.sup.(1&2) NA 6.50 NA 4.70 NA 3.40 1.90 1.38 14.2 60                         7.sup.(1&3) NA 2.80 NA 1.80 NA 4.10 0.70 0.44 8.3 NA                          8.sup.(2&3) NA 1.3 NA 0.8 NA 1.9 0.68 0.42 12.5 63                            9.sup.(1&2&3) NA 1.5 NA 0.6 NA 1.0 1.5 0.6 13.4 67                            Wild 408 1 423 1 232 1 1 1 41 28                                              Type ±136 ±0.33 ±47 ±0.11 ±67 ±0.28 ±0.33 ±0.26                                                          ±2 ±5              __________________________________________________________________________     NA--Not Available                                                             .sup.(1) Clot lysis test                                                      .sup.(2) ratio:                                                               ##STR6##                                                                      .sup.(3) Fibrindependent plasminogenolytic assay                              .sup.(4) Fibrinindependent plasminogenolytic assay                            .sup.(5) ratio:                                                               ##STR7##                                                                     7                                                                              .sup.(6) ratio:                                                               ##STR8##                                                                     8                                                                         

It is evident from the above results that substantial advantages can beachieved by making changes in the wild-type tPA amino acid sequence.Thus, not only can activity be increased, but at the same timesensitivity to plasminogen activator inhibitor can be decreased, so thatoverall a very substantial increase in effective activity can beachieved in vivo. Also, the enzyme can be made substantially morespecific in providing for enhanced fibrin dependence, so that it hassubstantially reduced activity in the absence of clots. Thus, one canadminister lower amounts of these polypeptides with plasminogenactivator activity, so as to minimize the level in the blood stream ofthe protein (or enzyme) and substantially diminish the undesirable sideeffects of tPA, while providing for increased activity against clots.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A human tissue plasminogen activator capable ofdissolving clots having lysine 277 substituted with another amino acid,and further comprising a deletion of from 3 to 25 amino acids from theC-terminus.
 2. A pharmaceutical composition comprising a human tissueplasminogen activator of claim
 1. 3. A nucleic acid molecule encoding ahuman tissue plasminogen activator of claim
 1. 4. A vector comprisingthe nucleic acid sequence of claim
 3. 5. A method of making the vectorof claim 4, comprising:(a) isolating said nucleic acid sequence; and (b)inserting said nucleic acid sequence into an expression vector such thatit would be expressed in an appropriate host cell.
 6. A host cellcomprising the nucleic acid sequence of claim
 3. 7. A method of making ahuman tissue plasminogen activator according to claim 1, comprising:(a)culturing a host cell comprising a nucleic acid encoding said humantissue plasminogen activator; and (b) isolating the protein.
 8. A humantissue plasminogen activator of claim 1, further comprising asubstitution of at least one of the group consisting of asparagine 117,asparagine 184 and asparagine 448 with another amino acid.
 9. Apharmaceutical composition comprising a human tissue plasminogenactivator of claim
 8. 10. A nucleic acid molecule encoding a humantissue plasminogen activator of claim
 8. 11. A vector comprising thenucleic acid sequence of claim
 10. 12. A method of making the vector ofclaim 11, comprising:(a) isolating said nucleic acid sequence; and (b)inserting said nucleic acid sequence into an expression vector such thatit would be expressed in an appropriate host cell.
 13. A host cellcomprising the nucleic acid sequence of claim
 10. 14. A method of makinga human tissue plasminogen activator according to claim 8,comprising:(a) culturing a host cell comprising a nucleic acid encodingsaid human tissue plasminogen activator; and (b) isolating the protein.